Nmda receptor antagonist formulation with reduced neurotoxicity

ABSTRACT

The present invention is directed to pharmaceutical compositions of effective amounts of NMDA receptor antagonists and preservative for the administration to a patient in need of effective analgesia and anesthesia. The compositions of the invention advantageously do not cause any significant neurotoxicity. The preferred NMDA receptor antagonist is ketamine. The preferred preservative is benzalkonium chloride.

FIELD OF THE INVENTION

The present invention is directed to reducing toxicity of an NMDAreceptor antagonist formulation. In particular, the invention isdirected to an NMDA receptor antagonist composition which isadministered for its analgesic and anesthetic effects, and which avoidssignificant neurotoxic side effects.

BACKGROUND OF THE INVENTION

An NMDA receptor is a postsynaptic, ionotropic receptor that isresponsive to, inter alia, the excitatory amino acids glutamate andglycine and the synthetic compound NMDA. The NMDA receptor controls theflow of both divalent and monovalent ions into the postsynaptic neuralcell through a receptor associated channel (Foster et al., Nature 1987,329:395-396; Mayer et al., Trends in Pharmacol. Sci. 1990, 11:254-260).Activation of the NMDA receptor has been shown to be the central eventwhich leads to excitotoxicity and neuronal death in many disease states,as well as a result of hypoxia and ischemia following head trauma,stroke and following cardiac arrest. The NMDA receptor has beenimplicated during development in specifying neuronal architecture andsynaptic connectivity, and may be involved in experience-dependentsynaptic modifications. In addition, NMDA receptors are also thought tobe involved in long term potentiation and central nervous systemdisorders.

It is known in the art that the NMDA receptor plays a major role in thesynaptic plasticity that underlies many higher cognitive functions, suchas memory acquisition, retention and learning, as well as in certainnociceptive pathways and in the perception of pain (Collingridge et al.,The NMDA Receptor, Oxford University Press, 1994). In addition, certainproperties of NMDA receptors suggest that they may be involved in theinformation-processing in the brain that underlies consciousness itself.

NMDA receptor antagonists are therapeutically valuable for a number ofreasons. In addition to anesthesia, certain NMDA receptor antagonistsconfer profound analgesia, a highly desirable component of generalanesthesia and sedation. Also, NMDA receptor antagonists areneuroprotective under many clinically relevant circumstances (includingneuropathic pain states, ischemia, brain trauma, and certain types ofconvulsions).

However, it is clear from the prior art that there are a number ofdrawbacks associated with current NMDA receptor antagonists. Theseinclude the production of involuntary movements, stimulation of thesympathetic nervous system, induction of neurotoxicity at high doses(which is pertinent since NMDA receptor antagonists have low potenciesas general anaesthetics), depression of the myocardium, andproconvulsions in some epileptogenic paradigms, e.g., “kindling” (Wlazet al., Eur. J. Neurosci. 1994; 6:1710-1719). There have beenconsiderable difficulties in developing new NMDA receptor antagoniststhat are able to cross the blood-brain barrier, which results in highereffective dosage requirements.

Commercially available NMDA antagonists have a wide variety of uses. Forexample, memantine provides rapid and enduring improvement in cognitive,psychological, social and motor impairments of dementia;dextromethorphan is used to relieve coughs; amantadine is an antiviralsubstance; and ketamine as an anesthetic agent. Certain opioids such asmethadone, dextropropoxyphene, and ketobemidone are also classified asNMDA antagonists. MK-801 (dizocilpine maleate) and phencyclidine are notcommercially used, and dextrophan, which is used commercially are otherexamples. However, a level of toxicity which accompanies theseantagonists has proven to be problematic.

There are numerous potential commercial applications for NMDA antagonistformulations without neurotoxicity in supervised medical practice.Indications include, but are not limited to, treatment of dementia,suppression of cough (antitussive), antiviral treatment, treatment ofinvoluntary muscle actions, antidepressant, suppression of addiction,and treatment of withdrawal. Ketamine, for example, can be used as ananalgesic for breakthrough pain, anesthesia and sedation. Additionalindications for ketamine include traumatic orthopedic injury pain,migraine pain, obstetrical use for end-stage labor pain, central pain,dental pain, and a host of additional conditions associated with acuteand chronic, moderate to severe pain.

More specifically, ketamine, an NMDA receptor antagonist, has been inclinical use for over twenty-five years as a dissociative anesthetic andhas demonstrated a wide margin of safety when used acutely as ananesthetic agent. Studies demonstrate the analgesic efficacy of ketaminein a variety of diverse indications including patient self-management ofpain (U.S. Pat. No. 6,248,789 and No. 5,543,434 to Weg), post-operativeanalgesia (Naguib et al., Can. Anaesth. Soc. J. 1986, 33:16;Dich-Nielsen et al., Acta Anaesthesiol. Scand. 1992, 36:583; Battacharyaet al., Ann. Acad. Med. Singapore 1994, 23:456), analgesia in emergencysettings for patients suffering from fractures and soft tissue injury(Hirlinger and Pfenninger, Anaesthsist 1987, 36:140), musculoskeletaltrauma (Gurnani et al., Anaesth. Intens. Care 1996, 24:32), wound careprocedures (Bookwalter, Plastic Surg. Nursing 1994, 14:43; Humphries etal., J. Burn Care Rehabil. 1997, 18:34), management of acute episodes ofneuropathic pain attributed to post-herpetic neuralgia (Eide et al.,Pain 1994, 58:347), phantom limb pain (Knox et al., Anaesth. Intens.Care 1995, 23:620), nociceptive orofacial pain (Mathisen et al., Pain1995, 61:215), and cancer pain (Mercadante et al., J. Pain SymptomManage. 1995, 10:564; Clark and Kalan, J. Pain Symptom. Manage. 1995,10:310; Fine, J. Pain Symptom Manage. 1999, 17:296; Lauretti et al.,Anesthesiology 1999, 90:1528). These studies describe the use ofketamine administered by a variety of routes including transnasal,parenteral, and oral.

There are conflicting results from studies evaluating the potential forketamine to cause neurotoxicity. Early in vitro studies examining themorphologic changes in cultured cells incubated with ketaminedemonstrated that the drug induced, to a varied extent, damage of themyelin sheath and degeneration of mitochondria into multilamellar bodiesin organotypic spinal cord slices derived from fetal rats (Shahar etal., Neurochem. Res. 1989, 14:1017). These apparent cytotoxic effects ofketamine were both dose-related and reversible. While no neurotoxiceffects of ketamine have been observed in primates or rabbits, spinalcord lesions have been reported in rats and monkeys (Ahuja, Br. J.Anaesth. 1983, 55:991; Malinovsky et al., Anesthesiology 1991, 75:91;Gebhardt, Anaesthesist 1994, 43(suppl. 2):S34). In addition, there isevidence of post-mortem histopathologic changes of subpial spinal cordvacuolation in a terminally ill cancer patient who received a continuousinfusion of intrathecal ketamine at a rate of 5 mg/day for a duration ofthree weeks (Karpinski et al., Pain 1997, 73:103). Based on thisfinding, it was concluded that intrathecal ketamine may cause vacuolarmyelopathy and that local vacuolation may be related to thelipophilicity of the drug. In addition, other studies have found thatNMDA receptor antagonists, as phencycladine, MK-801, tiletamine, andketamine cause neuronal vacuolization (Olney et al., Science 1989,244:1360).

The studies describing the potential neurotoxic effects of ketamine arelargely confined to administration of the drug by the intrathecal, orsubarachnoid, route. Intrathecal administration of drugs may producetoxic reactions such as demyelination, arrachnoditis, and vasucularchanges and necrosis.

According to standard practice, ketamine is usually employed containinga preservative. Studies comparing the neurotoxicologic profile ofpreservative-free ketamine to ketamine containing preservative(chlorobutanol or benzethonium chloride) yielded curious results.Experiments with baboons, monkeys, rabbits, and rats receiving between0.2 and 50 mg intrathecal ketamine with and without preservative failedto demonstrate histopathologic central nervous system lesionsattributable to the drug, but nonetheless detected a breach of the bloodbrain barrier that was attributable to the presence of preservative(Malinovsky et al., Anesthesiology 1993, 78:109; Karpinski et al., Pain1997, 73:103). The results were surprising since the combination of adrug with a preservative may also cause, or exacerbate, neurologicaldamage due to the preservative itself (Brock-Utne et al., S. A. Med. J.1982, 20:440). A further comparative study of multiple doseintrathecally administered preservative-free ketamine, ketaminecontaining the preservative benzethonium chloride, and benzethoniumchloride alone was performed in an attempt to resolve the apparentdiscrepancies in the animal models (Errando et al., Reg. Anesth and PainMed. 1999, 24:146). The results of this analysis demonstrated thatpreservative-free ketamine was without neurotoxic effect. However,ketamine with preservative produced minor changes to the spinal cord ofthe animals, and benzethonium chloride alone produced moderateneurotoxic effects (Errando et al., Reg. Anesth and Pain Med. 1999,24:146). The results of this study confirm the lack of apparentindependent neurotoxicity of ketamine and support the view thatpreservative-free ketamine is safe for intrathecal use in humans, evenfor repeated injections.

This observation was of limited value, however, since, while single-dosepreparations may not require preservatives, other substances require theaddition of preservatives to prevent or inhibit microbial growth andavoid spoilage of the preparation. Benzethonium chloride, a quaternaryammonium salt, is a common preservative similar to other cationicsurfactants. The animal models, noted previously, indicated that theaccompanying preservative, benzethonium chloride, and not ketamineitself, is the likely culprit mediating neurotoxicity in the anestheticformulation following intrathecal administration of the drugs. With theknown neurotoxic effects of this class of preservative, there remains aneed in the art for a safe and effective analgesic and anestheticformulation. The present invention addresses this need with a uniqueformulation which inhibits or diminishes the neurotoxicity.

SUMMARY OF THE INVENTION

It has now been discovered that henzalkonium quaternary ammoniumcompounds can be effectively used as preservatives for NMDA receptorantagonists which achieve the desired analgesic and anesthetic effects,without neurotoxic side effects. This discovery runs contrary toevidence of neurotoxicity associated with the administration of NMDAreceptor antagonists or antagonists with preservative. Thus, theinvention provides greater safety of prepared NMDA receptor antagonistformulations, which avoids the need to prepare preservative-freesolutions before every use.

The invention addresses the need in the art for effective preservativeswhich have no observable propensity to cause neurotoxicity, such asneuron vacuolation or degeneration. The discovery is particularlysurprising in that the benzalkonium compound is of the same class ofcompounds as the benzethonium compound which, as described above, hasdemonstrated neurotoxic side effects. Both are benzyl quaternarycompounds, which are cationic surfactants.

A particularly preferred NMDA receptor antagonist for use in theinvention is ketamine. A preferred benzalkonium compound is benzalkoniumchloride.

DETAILED DESCRIPTION

The present invention provides a formulation comprising a preservativeselected from the benzalkonium chloride quaternary ammonium salts, and atherapeutically effective dose of an NMDA receptor antagonist, i.e., adose effective to alleviate pain. The invention avoids the neurotoxicityof other preservatives and provides a formulation with reduced or closeto no neurotoxicity. The composition is administered for the analgesicor anesthetic effects without causing any significant neurotoxic sideeffects.

The use of intranasal ketamine has been studied as a safe and effectivetreatment for patients suffering from breakthrough pain. Becausebreakthrough pain can occur in chronic pain conditions, such as cancer,consideration was given to the possibility of neurotoxicity oftransnasal ketamine compositions containing benzethonium chloride. Thepresent invention is based on the discovery that ketamine formulations,as studied in rats, containing benzalkonium chloride as a preservativedemonstrated no observable effects in neuron vacuolation ordegeneration.

The present invention addresses the need in the art for pharmaceuticalcompositions comprising an NMDA receptor antagonist with an effectivepreservative having reduced neurotoxicity. Benzalkonium chloride is usedat relatively low concentrations (0.001-0.02%) and has optimal activitywhen pH is greater than 4, and at a pH up to 10, is stable at roomtemperature. Benzalkonium chloride is widely used as a preservative incommercial nasal sprays. Nasal irritation has been associated withchronic use of certain nasal products, and there have been isolatedreports of the ability of benzalkonium chloride to cause irritation tothe nasal mucosa. However, there appears to be no effect at theconcentration intended for use in the present invention's formulation(Kuboyama et al., J. Toxicol. Sci. 1997, 22:153).

The NMDA receptor antagonists used in the invention include, but are notlimited to ketamine, dextromethorphane, dextrorphan, methadone,dextropropoxyphene, ketohemidone, and phencycladine. In the preferredembodiment, the NMDA receptor antagonist is ketamine.

Other examples of antagonists include competitive and non-competitiveantagonists. The competitive NMDA antagonists include2-amino-7-phosphonoheptanoic acid (AP 7) and analogs;3-((±)2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP) andanalogs; (e)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid(CPPenes) and analogs;

cis-4-phosphonomethyl-2-piperidinecarboxylic acid (CGS 19755);DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 40115)enantiomers and analogs;S-α-amino-5-phosphonomethyl-[1,1′-biphenyl]-3-propanoic acid,E-2-amino-4-methyl-5-phosphono-3-pentenoic acid,E-2-amino-4-methyl-5-phosphono-3-pentenoic acid ethyl ester,cis-4-phosphonomethyl-2-piperidinecarboxylic acid,(R)-4-oxo-2-amino-5-phosphono-pentanoic acid,2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid,4-(phosphonomethyl)-DL-phenylglycine,4-(3-phosphonopropyl)-2-piperidinecarboxylic acid,2-(2-phosphonoethyl)-DL-phenylalanine,3-carboxy-5-(phosphonoethyl)-1,2,3,4-tetrahydroisoquinoline,3-carboxy-5-phosphono-1,2,3,4-tetrahydroisoquinoline,cis-DL-4[(1(2)H-tetrazol-5-yl)methyl]2-piperidinecarboxylic acid,cis-4-(3-phosphonoprop-1-enyl)-2-piperidinecarboxylic acid,E-2-amino-4-propyl-5-phosphono-3-pentenoic acid, phosphoricacid-4-(2-carboxy-piperidinyl) ester, and1-[4(4-chloro-α,α-dimethylbenzyl)-2-methoxyphenyl]-1,2,4-triazole-3-carboxylicacid amide. Noncompetitive NMDA antagonists include memantines and otheramantadine analogs; budipine and analogs; ifenprodil and analogs;antagonists of the glycine binding site kynurenic acid and analogs;1-hydroxy-3-aminopyrrolidin-2-one (HA-966) and analogs; polyamines suchas spermine and spermidine and analogs: inhibitors of the excitatoryamino acid synthesis.

When used as an anesthetic, i.e., to substantially eliminate allsensation, the dosage range is broadly from 1 mg/kg to 15 mg/kg, andpreferably from 1 to 4.5 mg/kg over a period of about 1 minute deliveredI.V. and 6.5 to 13 mg/kg via intramuscular injection.

On the other hand, when ketamine is used as an analgesic, i.e., toreduce or eliminate pain, the dosage range is broadly from 0.01 mg/kg to1 mg/kg, and preferably from 0.05 mg/kg to 0.7 mg/kg.

The preservatives used in the invention are benzalkonium chloridequaternary ammonium salts. These compounds have the formula:

wherein X is a halide. The phenyl ring may also have a Cl substitution.The R is an alkyl group having from 10 to 22 carbon atoms, preferably 12to 16 carbon atoms. The X may be bromide or iodide, but is preferablychloride. Most preferably R is a mixture containing C₁₂ and C₁₄ alkylgroups, and X is most preferably chloride, otherwise known asbenzalkonium chloride.

The amount of the preservative administered ranges from about 0.001% toabout 0.2% per unit dose, preferably from about 0.07% to about 0.14% perunit dose.

Other agents may be used in the invention, for example those that can beused for delivery including liposomes, microparticles (includingmicrospheres and microcapsules) and other release devices and forms thatprovide controlled, prolonged or pulsed, delivery or which enhancepassage through the blood brain barrier. Suitable pharmaceuticalcarriers, known to those skilled in the art, may also be used. These aredescribed in Remington's Pharmaceutical Sciences, 17th ed., MackPublishing Co., Easton, Pa., p. 1418 (1985), a standard reference textin this field, incorporated herein by reference. Antioxidizing agentssuch as sodium bisulfite, sodium sulfite, or ascorbic acid either aloneor combined are suitable stabilizing agents. Also used are citric acidand its salts and sodium EDTA.

Administration of the NMDA receptor antagonist can be by way of oral,transmucosal (buccal, nasal and rectal), transdermal, intramuscular, orintraocular route, or by parenteral administration. The parenteralroutes of administration include, but are not limited to, intravenous,intramuscular, intrathecal, epidural, intracerebroventricular,intradermal/intracutaneous, or subcutaneous injections. Other parenteralroutes may include intraarticular (into the joints), intrasynovial (ajoint-fluid area), intraspinal, intra-arterial, and intracardiac. Anytwo or more routes of administration can be combined, such asintravenous and transdermal.

As those skilled in the art recognize, many factors that modify theaction of the active substance herein will be taken into account by thetreating physician such as the age, body weight, sex, diet and conditionof the patient, the time of administration, the rate and route ofadministration, and so forth. Optimal dosages for a given set ofconditions can be ascertained by those skilled in the art usingconventional dosage determination tests in view of the experimental dataprovided herein.

The present invention is intended for use in animals. In a preferredembodiment, the invention is used with mammals. In another embodiment,the invention is directed to use in humans. The terms used in thisspecification generally have their ordinary meanings in the art, withinthe context of this invention and in the specific context where eachterm is used. Certain terms are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitioner indescribing the compositions and methods of the invention and how to makeand use them.

As used herein, the term “antagonist” refers to a compound that rendersthe active agent unavailable to produce a pharmacological effect. Inother words, the antagonist, itself, does not produce a particularpharmacological effect, but rather blocks the ability of an active agentto produce that effect. In a specific embodiment, the antagonistinteracts with the same receptor as the active agent and inhibits theinteraction of the active agent with the receptor. The term “antagonist”as used herein includes any compound that reduces the flow of cationsthrough an ionotropic receptor such as NMDA, i.e., a channel closer, andwhich has not been observed to increase the flow of cations through thesame receptor.

A “therapeutically effective amount” of a drug is an amount effective todemonstrate a desired activity of the drug. According to the instantinvention, in one embodiment a therapeutically effective amount ofketamine is an amount effective to alleviate, i.e., noticeably reduce,pain in a patient. In another embodiment, a therapeutically effectiveamount is an amount effective to enhance another pain therapy, e.g., apain medication such as a narcotic. in still another embodiment, it isan amount effective to induce anesthesia.

The term “neurotoxicity” as used herein refers to the level of neurondegeneration or necrosis, e.g., as measured by neuronal vacuolation orbehavioral changes after exposure to the NMDA antagonist composition.The NMDA receptor antagonist formulation of the present invention is onein which the neurotoxicity of the composition is reduced or close tonone. The term “close to none” means a level of neurotoxicity, if any,that cannot be detected by a particular assay method.

The term “nontoxic” as used herein shall be understood in a relativesense and is intended to designate any substance that has been approvedby the United States Food and Drug Administration (“FDA”) foradministration to humans or, in keeping with established criteria, issusceptible to approval by the FDA for administration to humans. Theterm “nontoxic” is also used herein to describe the NMDA receptorantagonists, or blockers, that are useful in the practice of the presentinvention from NMDA receptor antagonists such as MK-801 whose toxicitieseffectively preclude their therapeutic use.

As used herein, the term “pharmaceutically acceptable” refers to abiologically or pharmacologically compatible for in vivo use, andpreferably means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans.

The following Example(s) illustrate the invention, but are not limiting.

EXAMPLE 1 Randomized, Placebo-Controlled, Double Blind Study of theSafety and Efficacy of PMI-100 for the Treatment of Breakthrough Pain inPatients with Chronic Malignant Pain

This example evaluates the safety and efficacy of a ketaminehydrochloride formulation with preservative delivered through a nasalspray. Plasma levels were measured for the bioavailability and forcorrelating blood levels with analgesic effect.

Methods

The study is a randomized, multi-center, placebo-controlled,double-blind, crossover trial with 20 patients who had chronic malignantpain and a pattern of 2-7 episodes of daily breakthrough pain, despitetaking stable doses of analgesic medication. After an initial screeningvisit (visit 1), patients completed 2 study visits at least 48 hoursapart; one visit for treatment with a PMI-100 formulation (visit 2), anda second visit for treatment with placebo (visit 3). The PMI-100formulation is an aqueous intranasal ketamine formulation containing 10%(w/v) ketamine hydrochloride solution and 0.002% benzalkonium chloridesolution. The placebo control is an aqueous solution of 0.002%benzalkonium chloride solution alone.

When pain intensity at the onset of breakthrough pain episodes weregreater than or equal to 5 on the Numeric Pain Intensity Scale (LAPIS),patients self-administered 1-5 sprays (90 seconds apart, alternatingnostrils) of the PMI-100. If the episode was less than 5, the patientswere advised to wait for another episode. Treatment duration varieddepending on the number of sprays of study medication administered bythe patient.

The primary efficacy parameter was the difference between the average ofthe 9 post-treatment NPIS measurements and the baseline pre-treatmentNPIS pain measurement. Secondary efficacy parameters included: NPIS painmeasurements at each of 10 time points, the Investigator's opinion ofthe treatment response (rated as “good,” “fair,” or “poor”), and theproportion of subjects with at least a 40% reduction of the NPISmeasurement from baseline to the end of treatment; where “end oftreatment” was defined as the average of the 9 post-baseline NPISscores. Another parameter of interest was the proportion of subjects whotook rescue medication during the breakthrough pain episode.

Safety assessments consisted of monitoring and recording all adverse andserious adverse events, measurement of hematology and blood chemistryparameters, measurement of vital signs, measurement of nasal symptoms,including an assessment of the side effects utilizing a rating scale fordissociative anesthetics. The primary safety parameter was the resultsfrom the Side Effects Rating Scale for Dissociative Anesthetics. Thescale was administered immediately after the final NPIS rating was done(approximately 60 minutes after the first administration of studymedication) and then again 24 hours after administration of the studydrug.

Patients were asked to rate any of the side effects included in thescale that may have occurred since using the study medication. Sideeffects that were rated included: fatigue, dizziness, nausea, headache,feeling of unreality, changes in hearing, changes in vision, moodchange, generalized discomfort and hallucination. The degree of severityof each of these side effects was rated as: 0=No change; 1=weak,2=modest; 3=bothersome; 4=very bothersome. Other adverse events wererecorded separately. The Investigator recorded on the CRF whether eachadverse event was best described as UNRELATED, POSSIBLY RELATED,PROBABLY RELATED, DEFINITELY RELATED or of UNKNOWN association to thestudy medications.

Hematology [hemoglobin, hematocrit, PT, PTT, red blood cells (RBC),white blood cells (WBC) with differential and platelet count], bloodchemistry [sodium, CO₂, potassium, calcium, phosphorous, chloride,glucose, blood urea nitrogen, serum creatinine, serum uric acid, totalserum protein, serum albumin, total bilirubin, lactate dehydrogenase(LDH), liver function tests (AST, ALT, alkaline phosphatase)], weremeasured prior to the treatment and after the treatment at both Visits 2and 3.

Plasma levels of ketamine and its metabolites were measured at baseline,and at 2 minutes, 30 minutes, and 60 minutes following the last spray ofstudy medication for each patient.

Vital signs (body temperature, systolic blood pressure, diastolic bloodpressure, pulse oximetry and heart rate) were measured at Visit 1(screening), and pre-treatment, during treatment (at 10, 20, 40 and 60minutes) and post-treatment at Visits 2 and 3.

Nasal symptoms included the incidence of nasal pain, nasal congestion,sinus pain, sinus headaches, nosebleeds, change in smell, change intaste, runny nose, bad odor in nose, dry nose, postnasal drip, excesstearing and headaches.

The severity of break through pain episodes was rated using a NumericPain Intensity Scale (NPIS). The NPIS is a commonly used tool for theassessment of pain. The Side Effects Rating Scale for DissociativeAnesthetics provides an assessment of experiences likely to be inducedby near-anesthetic doses of drugs such as ketamine. The use of ketaminehydrochloride as an anesthetic agent has resulted in the occurrence ofpostoperative confusional states known as “emergence reactions” inapproximately 12 percent of patients. The psychological manifestationshave varied in severity, and in some cases have been accompanied byconfusion, excitement, and irrational behavior recalled by a fewpatients as an unpleasant experience. The duration ordinarily was nomore than a few hours; in a few cases, however, recurrences took placeup to 24 hours postoperatively. Thus the Side Effects Rating Scale forDissociative Anesthetics was administered immediately after treatment(approximately 60 minutes after the first dose of study medication) aswell as 24 hours after treatment with study medication.

Efficacy. The primary efficacy analysis consisted of a two-stagecrossover analysis of a summary measure of change in NPIS painmeasurement. The summary measure of change in NPIS pain measurement wascalculated by averaging the 9 post-treatment NPIS pain measurements, andthen subtracting the baseline NPIS pain measurement. The tests for adifferential carryover effect and period effect were performed usingWilcoxon rank sum statistics with exact critical values. If no carryoveror period effects were noted, treatment effect was assessed by comparingwithin patient differences using the Wilcoxon signed rank test. If asignificant carryover effect was detected, treatment effect was assessedusing Visit 2 data only, with the Wilcoxon rank sum test. The Wilcoxonsigned rank statistic and the Wilcoxon rank sum tests were obtainedusing exact critical values.

As a secondary analysis, if there was no evidence of a carry-over effectin the primary analysis, a Friedman's Repeated Measures Analysis ofVariance on Ranks test was used to compare the NPIS responses at the 10time points within each treatment group. If there was a statisticallysignificant carry-over effect (p<0.10) in the primary analysis, then theFriedman's test was calculated on Visit 2 data only. Secondary analysesalso included the proportion of subjects who exhibited at least a 40%reduction in NPIS from baseline to the end of treatment (where “end oftreatment” was defined as the average of the 9 post baseline NPISscores), with PMI-100 vs placebo. These proportions were compared usingthe exact version of McNemar's test for matched proportions.

The investigator's opinion of response to therapy under each treatmentregimen was summarized as a cross tabulation table. The exact version ofthe marginal homogeneity test was performed. Another parameter ofinterest was the proportion of evaluable subjects who chose to userescue medication during the breakthrough pain episode under the placebocondition, versus the proportion who chose to use it under the activetreatment condition. These figures were described by cross tabulationsand compared by the exact version of McNemar's test for matchedproportions.

Safety. Cross tabulations of the responses by treatment were presentedfor the

Side Effects Rating Scale for Dissociative Anesthetics evaluatedpost-evaluation and 24-hours post-evaluation. The exact version of themarginal homogeneity test was performed. The incidence of adverse eventsby treatment and by visit were displayed for all adverse events, seriousadverse events and associated adverse events. For routine laboratory andchemistry parameters and vital signs, continuous outcomes were analyzedusing the Wilcoxon Signed Rank test. In addition, summary statistics(sample size, mean, median, standard deviation, and range) were alsopresented for each treatment. The number and percent of patientsexperiencing specific nasal complaints under each treatment weretabulated.

Plasma levels of ketamine and its metabolites (norketamine anddehydronorketamine) were measured and listed for each patient atbaseline, 2 minutes, 30 minutes, and 60 minutes following the last sprayof study medication. 10 ml blood samples were drawn, and replaced with10 ml saline. The blood samples were drawn into a heparinized tube,which was subsequently gently inverted 10 to 15 times, and centrifugeduntil cells and plasma were separated. At least 5 ml plasma weretransferred into 7 ml storage vial, labeled, and frozen immediately at−20° C.

Results

Treatment of breakthrough pain with PMI-100 demonstrated significantreductions in pain intensity compared to treatment with placebo in thiscross-over study. Mean reduction from the baseline NPIS pain score was2.65 (mean summary measure of change over the 9 post-treatmentobservation time points) units in the PMI-100 treatment group comparedto 0.81 units in the placebo treatment group (p<0.0001). Fifteen of 20(75%) of patients administered the maximum 5 sprays of PMI-100.Statistically significant reductions in pain intensity compared toplacebo occurred as early as the 10 minute observation period, or 4minutes following administration of the 5 spray, and significancecontinued through the 60 minute observation time point. Nineteen of 20patients (95%) reported reduction in pain intensity within the 60 minuteobservation period after treatment with PMI-100, while only 10 of these20 patients (50%) reported a reduction after treatment with placebo.Nine patients (45%) had an average reduction in NPIS score of 40% orgreater following treatment with PMI-100 compared to only one patient(5%) following treatment with placebo (p=0.0078). Zero out of 20patients requested rescue medication during the 60-minute breakthroughpain episode observation period while 7 of 20 (35%) requested rescuemedications after treatment with placebo (p=0.0156).

The investigator's global assessment of the patient's general conditionfollowing study medication was “good” for 16 of 20 (80%) patients,irrespective of whether the patient was being treated with PMI-100 orplacebo. After PMI-100 treatment, 18 of 20 (90%) patients were assessedas “good”, one patient was assessed as “fair” and only one patient wasassessed as “poor”. Four patients were assessed as “poor” duringtreatment with placebo.

After treatment with PMI-100, 13 of 20 (65%) patients achieved a minimumNPIS score that was at least 40% lower than the pre-treatment score,compared with only 4 of 20 (20%) in the placebo group. The clinicalsignificance of this effect is further demonstrated by the observationthat 14 of 20 (70%) patients treated with PMI-100 achieved a NPIS scoreof 4 or less, the target used by most pain guidelines, and 11 of 20(55%) patients attained a minimum NPIS score of 2.2 or less while anequivalent reduction in NPIS score was achieved only in 2 of 20 (10%)patients after treatment with placebo. By contrast, after administeringtreatment with placebo, 10 of 20 (50%) patients reported no reduction inNPIS score during the 60 minute breakthrough pain episode while only 1patient reported no relief after treatment with PMI-100.

Equally impressive from a clinical perspective, is the rapidity of painrelief, with 15 of 20 (75%) achieving their minimum NPIS score within 25minutes, and 8 achieving their minimum NPIS score within 5-10 minutesfollowing treatment with PMI-100. Statistically significant pain reliefoccurred within 4 minutes of the delivery of the final intranasal sprayof PMI-100 (10 minutes from initial spray). See Table A below.

TABLE A NPIS Score-Change from Baseline over Time-ModifiedIntent-to-Treat Population Time after Start of Administration of StudyDrug (Minutes)^(a) 5 10 15 20 25 30 40 50 60 PMI-100 −1.50 −2.45 −2.79−2.51 −3.03 −2.98 −3.13 −2.86 −2.47 Placebo −0.56 −0.67 −0.83 −0.83−0.94 −0.96 −0.79 −0.77 −0.98 P-value^(b) 0.2114 0.0039 0.0007 0.00100.0003 0.0007 0.0001 0.0001 0.0037 ^(a)Administration of PMI-100 may nothave completed until 7.5 minutes after start of dosing ^(b)Wilcoxonsigned rank test comparing change between treatment groups

A statistically significant between-treatment difference in the meanchange of the NPIS score was observed as early as 10 minutes afterinitiation of treatment and persisted for remainder of the 60 minuteobservation period. For the 15 (75%) patients administering 5 sprays,statistically significant changes in NPIS score occurred as early as 4minutes following the final spray. The greatest improvements in the NPISscore occurred in the PMI-100 treated group between 25 and 40 minutesafter initiation of treatment. This corresponds with plasma levels ofketamine that were higher at the 30 minute post final spray observationpoint compared to the 2 minute and 60 minute post final sprayobservation points. Analyses were repeated for a per-protocolpopulation. Analyses suggested evidence of a period effect (p=0.0350)although subjects who were administered PMI-100 still had an advantageover those administered placebo at both Visit 2 (3.46 units vs. 1.20units, p=0.0734) and Visit 3 (2.05 units vs. 0.45 units). Results fromthe GEE analysis suggested that after adjusting for a period effect(p=0.0207), the reduction from baseline was on average 1.93 unitsgreater following treatment with PMI-100 than placebo (p=0.0029).

Based on the NPIS score reported, a rank was assigned to each of the 10visit time points for each patient. The maximum rank that could beassigned to a time point was 10. However, if a patient reported the sameNPIS score at more than one time point, the average rank would beassigned to each of those equivalent time points. The median of theranks of the 20 patients was then calculated at each time point. Themedian rank of the pre-treatment time point (time 0) was 9.5 forpatients following PMI-100 treatment and the 25^(th)-75^(th) percentilerange was 8.3 to 10.0. This would imply that the pain of maximumintensity was felt at time 0. Over time the median dropped and the rangeof the distribution shifted lower. At 60 minutes the median was 6.8 witha 25^(th)-75^(th) percentile range of 3.0 to 8.5. Friedman's testp-value of <0.0001 (0.0007 following treatment with placebo) suggeststhat there is a difference in the distribution of ranks over the 60minutes, and the contrast p-value of <0.0001 (0.0386 following treatmentwith placebo) indicates that there is a difference between time 0 andthe average of the post-treatment values.

Following the administration of study medication, the investigatorevaluated the patient's general condition as good, fair or poor. Sincethe investigator's global assessment was “good” following either PMI-100or placebo treatment for 16 of 20 (80%) patients, this assessment provednot to have discriminatory potential. This group included all 15patients evaluated at Site 01. Three patients were assessed as “poor”following treatment with placebo and required rescue medication duringthe breakthrough pain episode. Of the 20 patients in the study, onlypatient 164 from Site 03, received an assessment of “poor” aftertreatment with PMI-100. Patients in the per-protocol population wereassessed as “good” following treatment with both PMI-100 and placebo.

The number of patients that were able to attain a 40% reduction in NPISscore from the pre-treatment score to the mean of the 9 post-treatmentobservations was compared between treatment with PMI-100 and treatmentwith placebo. Nine of 20 (45%) patients attained a mean of the 9post-treatment observations that represented at least a 40% reductionfrom their pre-treatment NPIS score following treatment with PMI-100.These were patients 106, 107, 110, 112, 115, and 117 from Site 01 and162,163 and 165 from Site 03. Patient 110 from Site 01 also attained a40% reduction following treatment with placebo. This treatmentdifference in ability to attain a greater than 40% reduction in NPISscore was shown to be statistically significant compared to placebo(p=0.0078) using McNemar's test for paired data.

Use of rescue medication during the 60-minute breakthrough painevaluation period was compared between patients being treated withPMI-100 and those being treated with placebo. Zero of 20 patientsrequired rescue medication during the breakthrough period when treatedwith PMI-100, compared to 7 of 20 patients that required rescuemedication during the breakthrough period after treatment with placebo(p=0.0156).

Efficacy Conclusions. The results of the study indicate that intranasalketamine hydrochloride (PMI-100), administered at doses ranging from 10mg to 50 mg, provides rapid, meaningful pain relief during intensebreakthrough pain episodes that otherwise are poorly managed. Placebocontrol had no effect in alleviating pain. The onset of statisticallysignificant reductions in pain intensity after treatment with PMI-100was within 10 minutes of the first spray, and within 4 minutes forpatients who administered the full 5 sprays. Furthermore, 75% ofpatients treated with PMI-100 achieved their minimum NPIS score within25 minutes of administration. The magnitude of the average decrease inpain intensity compared to placebo was statistically significant overthe 60 minute observation period, and a statistically significant numberof patients averaged a >40% reduction in their NPIS scores. Thetime-specific significant reductions in pain intensity after treatmentwith PMI-100 started at 10 minutes and continued through the 60 minuteobservation point. The efficacy data presented here is clinicallysignificant. Zero of 20 patients required use of rescue medications inthe 60 minutes following administration of PMI-100, representing thepotential for lower daily opioid use. Although patients were notrequired to administer 5 sprays, or 50 mg, of PMI-100 the majority (75%)did, indicating that the therapeutic dose is possibly towards the higherend of the range. Patients who delivered less than 5 sprays did indicatesubstantial reductions in pain intensity however, which supports aself-titrating approach might be necessary with this test product.

Safety Evaluation. Using the Rating Scale for Dissociative Anesthetics,which was a retrospective questionnaire administered to each patient 60minutes post-study drug administration, possible dissociative-type sideeffects were evaluated. The questionnaire was administered again 24hours after study drug administration to evaluate any lingering effectsof intranasal ketamine.

Overall there were few dissociative side effects reported, and themajority of effects were mild to moderate in severity and had resolvedby the time the questionnaire had been administered 60 minutes afterdosing. Nine of 20 (45%) patients reported some type of dissociativeeffect following treatment with PMI-l00 compared to only 1 (5%) patientafter treatment with placebo. The most commonly reported effectsreported on the questionnaire after treatment with PMI-100 were fatigue(7 patients), dizziness (4 patients), feeling of unreality (4 patients),and changes in vision (2 patients). Of these commonly reported effects,less than 10% of the treatment group indicated that they were“bothersome” or “very bothersome” in nature. Only one patient (5%)indicated a “general feeling of discomfort” after treatment withPMI-100. With the exception of 2 patients with fatigue, and one patientwith nausea, there were no side effects reported on the 24 hourpost-drug administration dissociative side effects questionnaire. Therewere no reports of hallucinations following treatment with intranasalPMI-100, nor were any interventions with benzodiazipines required.

The majority of bothersome and very bothersome dissociative effectsreported were experienced by three patients, all of whom administeredthe maximum 5 sprays of PMI-100: 1) Patient 165 experienced verybothersome dizziness and feeling of unreality post-evaluation. Thepatient also experienced dizziness and headache during placeboadministration. This patient also had a fluctuation of blood pressure,with a pre-episode blood pressure of 142/86. Twenty minutes into thebreakthrough pain episode, the patient's blood pressure rose to 169/88.At post-evaluation, the patient's blood pressure was 103/53. 2) Patient117 experienced 7 side effects post-evaluation which included fatigue(bothersome), dizziness (bothersome), feeling of unreality (moderate),change in hearing (moderate), change in vision (mild), mood change(moderate), and generalized discomfort (bothersome). 3) Patient 105experienced fatigue (bothersome), feeling of unreality (bothersome), anda change in vision (moderate) at post-evaluation. It should be notedthat this patient had a medical history significant for blurry vision inthe past, so this is possibly not related to the drug.

No patients, following administration of either PMI-100 or placebo,experienced dizziness 24 hours post evaluation. No patients, followingadministration of either PMI-100 or placebo, experienced headachepost-evaluation, or 24 hours post-evaluation. No patients, followingadministration of either PMI-100 or placebo, experienced a feeling ofunreality 24 hours post-evaluation. No patients, followingadministration of either PMI-100 or placebo, reported changes in hearing24 hours post-evaluation. No patients, following administration ofeither PMI-100 or placebo, experienced changes in vision 24 hourspost-evaluation. No patients, following administration of either PMI-100or placebo, reported mood change 24 hours post-evaluation. No patients,following administration of either PMI-100 or placebo, experiencedgeneralized discomfort 24 hours post -valuation.

A pre- and post-treatment nasal symptom assessment was performed as partof the safety assessment of PMI-100 and placebo. The nasal spray, at aconcentration of 100 mg/ml, and a dose of 10 to 50 mg (one to 5 sprays),was very well tolerated with few effects noted on the nasal symptomexam. A change in taste that was not present pre-treatment was reportedfor 3 patients (#162, 163 and 165 from Site 03) following treatment withPMI-100. Nasal congestion, sinus pain, runny nose and post-nasal dripwere also reported nasal symptoms. These symptoms were all presentpre-treatment and may or may not have been observed post-treatment.

A total of 10 patients experienced adverse events after treatment witheither PMI-100 or placebo. All but 4 treatment-emergent adverse eventswere considered to be associated treatment emergent adverse events,those events deemed by the investigator as either possibly, probably, ordefinitely related to the study medication. Six of 20 (30%) patientsthat received PMI-100 experienced an associated treatment-emergentadverse event categorized under the body system of nervous systemdisorders. Two of 20 (10%) patients that received placebo reportedadverse events that fell into this category.

Two of 20 (10%) patients experienced a moderate elevation in bloodpressure within 15 minutes following administration of the full dose (50mg) of PMI-100. Patient 164 had an initial pre-episode blood pressure of165/69 prior to treatment with PMI-100. During this breakthrough painepisode, the patient's blood pressure rose to 212/88. Approximately60-minutes after the first spray of PMI-100 the patient's blood pressurewas 191/84. This adverse event fell under the body system“investigations” and had a preferred term of “blood pressure increased.”This patient's blood pressure also rose during the breakthrough painepisode that was treated with placebo. The patient had a pre-episodeblood pressure of 155/70. At 60-minutes, the patients' blood pressurehad risen to 187/85, and then dropped back down to 154/73 atpost-evaluation. Patient 165 also experienced an increase in bloodpressure during the breakthrough pain episode that was treated withPMI-100. The patient's pre-episode blood pressure was 142/86. At20-minutes, the patient's blood pressure had risen to 169/88. Atpost-episode evaluation, the patient's blood pressure had dropped to103/53. This adverse event fell under the body system of “vasculardisorders” and had a preferred term of “hypertension NOS.”

Only 4 treatment-emergent adverse events were considered not associatedto the study medication, 2 occurred following treatment with placebo(mild laceration, nausea) and 2 following treatment with PMI-100(pyrexia, nasal congestion). No serious treatment emergent adverseevents occurred during the study. One patient (165) from study Site 03experienced a serious adverse event during the screening phase of thestudy. Patient 165 experienced intractable vomiting and was hospitalizeddue to this serious adverse event. This event was considered unrelatedto the study medication, since the patient had not received any studymedication at the time of the event.

Safety Conclusions. Intranasal ketamine (PMI-100) was well toleratedwith no serious adverse events, deaths, or treatment-related drop-outson study. The majority of adverse events were mild in severity andtransient in nature, with “change in taste” or “taste disturbance” beingthe most frequently reported effect after treatment with PMI-100. Duringtreatment with either PMI-100 or placebo, safety was assessed throughthe reporting of specific side effects using the Side Effects RatingScale for Dissociative Anesthetics, the existence of nasal symptomsassessed during a nasal exam, the monitoring of adverse events and themeasurement of vital signs, routine hematology and blood chemistryresults.

The results from the retrospective solicitation of possible dissociativeside-effects indicated that mild, transient fatigue, dizziness, and afeeling of unreality were the most commonly chosen items from thequestionnaire. Both dizziness and feelings of unreality were reported by4 out of the 20 subjects treated with PMI-100, while fatigue wasreported by 9/20 patients after treatment with PMI-100. The majority ofdissociative effects following treatment with PMI-100 were experiencedby 3 patients. The twenty-four hour post study dissociative side effectquestionnaire indicated no clinically significant residual effects aftertreatment with either PMI-100 or placebo, with 2 reports of fatigue, andone report of nausea. There were no hallucinations reported as a resultof treatment from either PMI-100, or placebo, and no interventions withbenzodiazipines were required.

Two patients experienced moderate elevations in blood pressure within 15minutes of treatment with PMI-100, which is consistent with knowneffects of ketamine. Both episodes resolved spontaneously with nosequelae. Vital signs were monitored throughout the study and there wereno clinically significant changes. No abnormal laboratory values ofclinical significance were reported that could not be attributed to aprevious condition.

The overall safety of treatment with PMI-100 for the treatment ofbreakthrough pain was shown to be similar to treatment with placebo.Although patients experienced more specific side effects after treatmentwith PMI-100 than after placebo, these transient side effects were mildand might be considered inconveniences rather than obstacles to ketaminetreatment.

Discussion and Overall Conclusions

During this 2 site, 2 phase crossover study, patients used an NumericPain Intensity Scale to rate their response to self-administeredintranasal ketamine hydrochloride (PMI-100) or placebo for the relief ofpain of >5 in intensity in 2 separate breakthrough pain episodes. All ofthese patients were opioid experienced, with daily,around-the-clockopioid regimens equivalent to at least 60 mg/daymorphine for the treatment of chronic pain, and additional, short-actingopioids equivalent to at least 5 mg morphine for breakthrough painepisodes. The primary endpoint of the study was to compare the averagereduction in pain intensity during a breakthrough pain episode aftertreatment with PMI-100 compared to placebo. The results of the studyindicate that 1 to 5 sprays (10 to 50 mg) of self-administeredintranasal PMI-100 compared to placebo demonstrated a highlystatistically significant (p<0.0001) reduction in average pain intensityover the 60 minute observation period. The average number of spraysadministered was 4.65 for PMI-100, indicating a therapeutic effecttowards the higher end of the dose range of 10 to 50 mg. All time pointsfrom 10 minutes through 60 minutes showed a statistically significantreduction in pain intensity for PMI-100 compared to placebo. Thestatistically significant reduction in pain intensity within 10 minutesof the initial spray of PMI-100, and 4 minutes of administration of 5sprays of PMI-100 is clinically relevant. In addition, patients requiredsignificantly more additional rescue medication for breakthrough painepisodes treated with placebo than for episodes treated with PMI-100.Considered to be a clinically relevant reduction in pain intensity frombaseline, significantly more patients achieved a 40% or greater overallreduction in pain intensity after treatment with PMI-100 compared totreatment with placebo.

Intranasal administration of PMI-100 for the treatment of breakthroughpain was well-tolerated, with no serious adverse events, deaths,treatment-related drop outs, or clinically significant side effects. Thenasal spray was well tolerated with a change in taste being the mostfrequently reported effect after treatment with PMI-100 at a rate of3/20 (15%) patients. A review of the literature published regarding theuse of intranasal ketamine reveals little about the possibility ofunpleasant dissociative side effects at sub-anesthetic dose levels ofketamine. In order to understand fully the potential for dissociativeeffects following intranasal treatment with PMI-100, a retrospectivequestionnaire was designed for this study that covered the range ofpsychotomimetic effects that could potentially occur. As with mostsolicitation tools, the prevalence of reported effects becomes somewhatinflated when providing a “menu” of items to choose from. The results ofthe side effect questionnaire indicate that the majority of effects weremild and transient, and few patients experienced troublesomedisorientation or feelings of unreality, and no patients experiencedhallucination or required intervention with benzodiazipines. Given theoverall unpleasantness of an intense episode of breakthrough paindespite the use of around-the-clock opioids, the transient effects ofketamine appear to be of no consequence in light of the pain-relievingqualities experienced after treatment during this study.

In conclusion, this randomized, placebo-controlled, double blind, pilotstudy in 20 patients has demonstrated that intranasal ketamine is ahighly efficacious treatment for malignant and non-malignantbreakthrough pain and shows a large margin of safety in patients onchronic opioid therapy. The profile of this experimental treatment asdemonstrated from this study is one of rapid onset, transnasalabsorption, possible titrateability, ease of use, and acceptance bypatients and for these reasons makes it ideally suited for the treatmentof breakthrough pain.

EXAMPLE 2 An Acute Subcutaneous Neurotoxicity Study in Rats with PMI-100

This Example evaluates the neurotoxicity of a formulation of ketaminehydrochloride (referred to hereafter as “PMI-100”) in rats following asingle subcutaneous injection. PMI-100 is a formulation containing 100mgketamine/ml and 0.002% benzalkonium chloride. The findings are based onthe level of neuronal vacuolation.

Materials and Methods

This study included 160 rats with 16 male and 16 female rats in each ofthe following five treatment groups: Group 1 was given sterile water forinjection (control); Group 2 was given 4 mg/kg PMI-100; Group 3 wasgiven 15 mg/kg PMI-100; Group 4 was given 60 mg/kg PMI-100; and Group 5was given 0.5 mg/kg MK-801. Four rats of each sex in each of these fivegroups were allocated to four study subgroups (Subgroups A, B, C, andD). The rats in Subgroup A were necropsied approximately six hourspost-dose. Those in Subgroup B were necropsied approximately 24 hourspost-dose. The rats in Subgroup C were necropsied approximately 72 hourspost-dose, and those in Subgroup D were necropsied 14 days post-dose.Table A illustrates the specific criteria of each group and subgroup.Table B illustrates the details of the subgroups and the stains employedfor evaluating the brain sections.

TABLE A Experimental Study Design - Acute Neurotoxicity Study No. ofApproximate Dose Dose Animals Dose Dose Level Conc. Volume Group MaleFemale Material (mg/kg) (mg/mL) (μL/kg) 1 16 16 Vehicle 0 0 600 2 16 16PMI-100 4 100 50 3 16 16 PMI-100 15 100 150 4 16 16 PMI-100 60 100 600 516 16 MK-801 0.5 5 100

TABLE B Subgroup Information-Acute Neurotoxicity Study Designation StudyPurpose Subgroup A Animals euthanized at ~6 hours post-dose (day 0)primarily for evaluation of neuronal vacuolation (H&E staining) SubgroupB Animals euthanized at ~24 hours post-dose (day 1) primarily forevaluation of neuronal vacuolation (H&E staining) and neuronal necrosis(Fluro-jade/DAPI staining). Subgroup C Animals euthanized at ~72 hourspost-dose (day 3) primarily for evaluation of neuronal necrosis (CupricSilver staining). Subgroup D Functional Observation Battery, (“FOB”) andlearning/memory evaluations conducted on this Subgroup. Animalseuthanized at 14 days post-dose (day 14) primarily for evaluation ofneuronal necrosis (Cupric Silver staining).

Histotechnology Procedures. Prior to necropsy, the rats wereanesthetized and then subjected to intracardiac perfusion for optimalfixation. The brains of rats in Subgroups C and D were removed andimbedded in a gelatin matrix (16 brains in each block), frozen, seriallysectioned at 40 micrometers. Representative step sections (between 33and 34 for each rat) were stained with a cupric silver stain accordingto the method of de Olmos, which is incorporated herein by reference(Fix et al., Toxicol. Pathol. 1996, 24:291-304; Switzer, R. C., New YorkAcad. Sci. 1993, 679:341-348; Switzer, R. C., Toxicol. Pathol. 2000,28:70-83). Of the 33-34 sections present for each rat brain,approximately 17 included the posterior cingulate and retrosplenialcortices.

The brains from the rats in Subgroups A and B were completely sliced ina standardized fashion to yield nine coronal sections, with each coronalslice being between 2-3 millimeters in thickness. Of these nine coronalsections, one included the posterior cingulate cortex, while threepassed through the retrosplenial cortex. Two sagittal sections of theolfactory bulbs were also prepared. The coronal brain slices were placedanterior face down (the olfactory bulbs medial surface down) withintissue cassettes, processed to paraffin with a Citadel® tissue processor(Shandon Lipshaw), and embedded in paraffin following standardizedprocedures. The paraffin blocks were sectioned at a thickness ofapproximately 5 micrometers using a rotary microtome. All brain sectionsfrom the rats in Subgroups A and B were stained with hematoxylin andeosin (H&E). In addition, duplicate sections of brain from the rats inSubgroup B were also stained with Fluoro-Jade B, using DAPI (4′,6-diamidino-2-phenylindole) as a counterstain. The Fluoro-Jade Bprocedure has been described previously in Schmued, L C and Hopkins, KJ., Brain Research 2000, 874:123-130 and in Schmued, L C and Hopkins, KJ., Toxicologic Pathology 2000, 28:91-99, which are both incorporatedherein by reference.

The cupric silver technique is the most sensitive stain fordemonstrating degenerative neurons (i.e., of the three stains used inthis study). The H&E stain is the least sensitive (or least specific)stain, with the sensitivity of the Fluoro-Jade B stain lying in betweenthat of the H&E and cupric silver stain. However, the cupric silverstain is frequently also characterized by non-specific staining that maybe confused with bone fide neuoronal degeneration. In this study, forexample, the lateral hypothalamic area frequently contained well-stainedfragmented axons that were present with equal frequency in the controland treated rats. This staining pattern was interpreted as beingartifactual in nature and, therefore, was not documented. Similarly,small numbers of axons with a fragmented or “beaded” appearance werefrequently found in other regions of the brain. These were notdocumented unless two or more stained axons were present in relativelyclose proximity (e.g. within one medium power field). The presence oftwo or three such stained axons would have received a grade of “minimal”for axonal degeneration. However, in the case of degenerating neurons,even one darkly-stained shrunken neuron would have received a minimalgrade for neuronal degeneration. (Note the “neuron degeneration” ratherthan “neuron necrosis” was used for the cupric silver-stained sections,because it was often not possible to definitively identify a necroticphenotype with this stain.) Darkly-stained neurons without a necroticphenotype (e.g. with well preserved nuclear detail) were considered torepresent artifactual “dark neurons” and were not documented.Darkly-stained (presumably degenerative) astrocytes within cupricsilver-stained sections were also not documented, these cells beingpresent in approximately equal numbers within both control and treatedrat brains.

Dark granular staining of scattered glomeruli within the olfactory bulbsof rats is quite common in sections stained with the cupric silvertechnique and is considered to represent normal backgrounddegeneration/remodeling. Although a greater frequency of this stainingis evident within treated rats in Subgroup C versus controls, this isnot considered to be of biologic significance based on past experiencewith this stain and this particular staining pattern. Because numerousother studies that have been evaluated have shown mild to moderatedegrees of olfactory glomerular degeneration within 100% of controlanimals, the inter-group differences in this particular study arethought to be the result of two factors: (1) that the control andtreated rat brains were embedded in different blocks (i.e., with all 16control group brains in Subgroups C and D having been blocked,together); and (2) the fact that the degree of this staining variesconsiderably from section to section.

Minimal degrees of axonal degeneration within cupric silver-stainedsections (i.e., usually two or three axons within one intermediate powerfield) or of neuron degeneration (usually one neuron within oneintermediate power field) can be overlooked as representing backgroundchange. Therefore, only neuron degeneration graded as being mild orgreater in degree within cupric silver-stained sections was consideredto be of biologic significance. Such degrees of degeneration werepresent only in the MK-801-treated rats.

Microscopic Evaluations. All slides were examined in “blinded” fashion(i.e., without knowledge of treatment group assignment). The labelspresent on the microscope slides for Subgroups A and B included thetreatment group designation and were, therefore, covered with opaquetape and the animal identifications replaced with letter codes. Nolabels were present on the cupric silver-stained slides, with only a keybeing present inside the slide box cover to indicate the animal numberfor each of the sixteen sections present on each slide. Microscopicfindings were hand-recorded by the pathologist onto individual animalwork sheets, with one sheet/animal being present for each set of H&E,Fluoro-Jade B, and cupric silver-stained slides. Diagnoses were eitherhand-written or numbers were used to indicate diagnoses present within astandard list (list included with raw data). All observations were alsogiven one of five grades of severity (minimal, mild, moderate, marked,or severe). Distribution patterns of focal, multifocal, or diffuse werealso assigned to any microscopic observations.

After removing the tape to “unblind” the animal numbers and groupassignments, the hand-recorded data were entered into a PC-basedcomputer program (GLPATH available from Great Laboratory Programs®). Thecomputer protocol for this study was set up to include only 31representative neuroanatomic regions, these having been selected basedboth on the potential for lesions to develop in these regions and toindicate the levels of brain that were examined. However, allareas/structures within every section were examined, not just theposterior cingulate and retrosplenial cortices.

All 33-34 cupric silver-stained sections per rat (Subgroups C and D)were also examined microscopically.

Results and Discussion

The microscopic findings for this study are discussed by subgroup.

Subgroup A. The rats in Subroup A had been euthanized approximately sixhours post-injection. Only H&E-stained brain sections were examined fromthese rats, primarily to look for evidence of neuron cytoplasmicvacuolation (especially within the posterior cingulate and retrosplenialcortices) typical of that seen following treatment with NMDA receptorantagonists such as MK-801 (Fix et al., Toxicol. Pathol. 1996,24:291-304; Fix et al., Experimental Neurology 1993, 123:204-215). Thistypical pattern of neuron vacuolation within the retrosplenial cortex,primarily involving neurons within Layers 2 and 3 of the cortex, waslimited to the MK-801-treated rats in this study and was most prominentin the female rats. Only one male rat treated with MK-801 had suchvacuolation, and this vacuolation was only minimal in degree. While thissame male rat (#4876) also had a minimal degree of neuron necrosiswithin the retrosplenial cortex, it is not certain that this neuronnecrosis was the result of the MK-801.

In contrast to the male rats, all four of the Subgroup A female ratstreated with MK-801 had mild to moderate degrees of neuronal cytoplasmicvacuolation within the retrosplenial cortex. In addition, twoMK-801-treated female rats had minimal to mild degrees of vacuolationwithin the piriform cortex. While none of the PMI-100-treated rats hadevidence of neuronal cytoplasmic vacuolation within any brain section,all four of the high dose group (60 mg/kg) PMI-100-treated rats hadminimal to mild degrees of vacuolation within the molecular layer(Layer 1) of the retrosplenial cortex. This vacuolation may indicate thepresence of swollen apical dendrites (i.e., from neurons present deeperwithin the retrosplenial cortex) or swollen axonal terminals belongingto neurons projecting to this region. However, there was no evidence atlater stages of any associated neuron degeneration (see discussion forSubgroups B-D, below). Also, a similar pattern of molecular layervacuolation was not present within any of the rats injected with MK-801.

In all of the Subgroups, there were minimal degrees of axonaldegeneration within the trapezoid body of the brainstem. However, thisrepresents a common background lesion in rats and was found in equaldegrees in Control group rats and was not treatment-related.

Subgroup B. The rats in Subgroup B had been euthanized approximately 24hours after receiving their single injections. Both H&E and Fluoro-JadeB-stained brain sections were examined from these rats, primarily todetect any residual vacuolation and/or early evidence of neuronalnecrosis within the posterior cingulate and retrosplenial cortices. InSubgroup B, one male and several female MK-801-treated rats had minimalto mild degrees of neuron necrosis within the piriform cortex that weredetected primarily within the Fluoro-Jade B-stained sections. None ofthe male rats in Subgroup B (i.e., even those treated with MK-801) hadevidence of treatment-related neuron degeneration or necrosis. However,three of the four female rats treated with MK-801 had minimal to milddegrees of neuron necrosis within the retrosplenial cortex. The fourthfemale rat treated with MK-801 was classified as having minimal “neurondegeneration” (rather than necrosis) within its retrosplenial cortex,because it was not certain whether the microscopic changes representedbasophilic neuron artifact or neuron necrosis (e.g. see Garman, R. H.,Toxicol. Pathol. 1990, 18:149-153.). No rats in Subgroup B that had beeninjected with PMI-100 had any microscopic evidence of neuron necrosiswithin any section of brain.

One male control group rat in Subgroup B had evidence of unilateraloptic tract degeneration that involved the optic nerve, optic tract, andsuperior colliculus. This is a common sporadic lesion of rats that isusually unilateral but occasionally bilateral in distribution (Shibuyaet al. J Vet Med Sci 1993, 55:905-912.). This lesion was also detectedin rats from Subgroups C and D and is not treatment related in thisstudy. As in Subgroup A, numerous rats in Subgroup B had evidence ofminimal to mild axonal degeneration within the trapezoid body, but thiswas also not a treatment-related finding in this study.

Subgroup C. The rats in Subroup C had been euthanized approximately 72hours after receiving their injections, with the brains from these ratshaving been step-sectioned and stained by the cupric silver technique todetect the presence of neuronal degeneration within the posteriorcingulate and retrosplenial cortices, as well as elsewhere within thebrain. There were between 33 and 34 sections from each brain.

One MK-801-treated male rat had mild neuron degeneration within thefrontal cortex. Two male MK-801-treated rats had mild to moderatedegrees of neuron degeneration within the retrosplenial cortex.

Mild to marked degrees of neuron degeneration within female rats werealso limited to the rats in the MK-801-treatment group, with noPMI-100-treated rats having similar evidence of neuron degeneration. Inthe female rats treated with MK-801, such neuron degeneration waspresent in the piriform cortex and retrosplenial cortex. Two female ratstreated with MK-801 also had evidence of mild terminal degenerationwithin the stratum lacunosum moleculare of the hippocampus. Suchterminal degeneration within the hippocampus was not seen inPMI110-treated rats.

One control group male rat in Subgroup C had marked unilateral axonaldegeneration within the optic nerve and optic tract indicative ofunilateral optic tract degeneration. A similar case of spontaneous optictract degeneration was present in one female rat in Group 2 (4 mg/kgPMI-100).

Subgroup D. The rats in Subgroup D had been euthanized 14 days afterreceiving their injections, with the brains from these rats beingstep-sectioned and stained by the cupric silver technique to detect thepresence later stages of neuronal necrosis within the posteriorcingulate and retrosplenial cortices (as well as elsewhere within thebrain). As in Subgroup C, the female rats treated with MK-801 were mostprominently affected. (Note that, as with Subgroup C, olfactory bulbglomerular degeneration and minimal degrees of neuron degeneration arenot considered to be of any biologic significance.) Mild neurondegeneration was found within the retrosplenial cortex of oneMK-801-treated male rat. Neuron degeneration graded as being either“mild” or “moderate” was found in the retrosplenial cortex of all fourof the MK-801-treated female rats in Subgroup D. However, as in theother subgroups on this study, no such degeneration was found within anyof the brain sections from rats treated with the PMI-100. All four ofthe MK-801-treated female rats in Subgroup D also had mild synapticterminal degeneration within the stratum lacunosum moleculare of thehippocampus. However, such degeneration was not found within any brainsections from PMI-100-treated rats.

The brain sections from one male rat in Group 2 (4 mg/kg PMI-100) werecharacterized by spontaneous optic tract degeneration, a conditionalready discussed as representing a spontaneous “background lesion.”

CONCLUSIONS

Blinded microscopic evaluations were performed on brain sections fromrats given one subcutaneous injection of either sterile water, MK-801 orthe PMI-100 formulation of ketamine hydrochloride. Sections of thebrains were examined from 4 female and 4 male rats at each of 6 hours,24 hours, 72 hours, and 14 days post-injection, with these sectionshaving been stained with either H&E, Fluoro-Jade B, or the cupric silvertechnique. Neuron vacuolation and degeneration within the retrosplenialcortex of the type typically seen with NMDA receptor antagonists waslimited to the rats injected with MK-801 and was most prominent in thefemale rats. Although these alterations were not present in the ratsinjected with any of the three dosages of PMI-100 used in this study(viz. 4, 15, or 60 mg/kg), the female rats in the high dose PMI-100group did have minimal to mild degrees of vacuolation within themolecular layer (Layer 1) of the retrosplenial cortex at 6 hours (withinthe H&E-stained sections). However, no evidence of neuron degenerationwas seen in the females from this dose group at later time points(within sections stained either with H&E, Fluoro-Jade B, or the cupricsilver technique). It is likely that the molecular layer vacuolation mayrepresent transient swelling within dendritic or axonal terminals.

This study had no-observable-effect level for the PMI-100 formulation ofketamine hydrochloride in this particular study of 60 mg/kg for the malerats and 15 mg/kg for the female rats. The ketamine hydrochlorideformulation containing benzalkonium chloride also did not produce anypermanent degenerative alterations.

EXAMPLE 3 A 28-Day Subcutaneous Neurotoxicity Study in Rats in PMI-100

The objective of this study was to evaluate the potential neurotoxicityof the test article in the rat following multiple subcutaneousinjections of the PMI-100 formulation containing 10 mg/kg (10% w/v)ketamine hydrochloride and 0.002% benzalkonium chloride solution. Theformulation was given once daily over a 28-day period.

Methods

192 rats were distributed across five treatment groups as indicated inTable A. Summary information, including dosing information and thestains employed for evaluating the brain sections, are presented in TextTables A and B, below.

TABLE A Experimental Study Design - Acute Neurotoxicity Study No. ofApproximate Dose Dose Animals Dose Dose Level Conc. Volume Group MaleFemale Material (mg/kg) (mg/mL) (uL/kg) 1 20 20 Vehicle 0 0 600 2 20 20PMI-100 4 100 40 3 20 20 PMI-100 15 100 150 4 20 20 PMI-100 60 100 600 516 16 MK-801 0.3-0.5 3-5 100

TABLE B Subgroup Information-Acute Neurotoxicity Study SubgroupDesignation Study Purpose Subgroup A Animals euthanized at ~6 hourspost-dose (day 27) primarily for evaluation of neuronal vacuolation (H&Estaining). Subgroup B Animals euthanized at ~24 hours post-dose (day 28)primarily for evaluation of neuronal vacuolation (H&E staining) andneuronal necrosis (Fluro-Jade/DAPI staining). Subgroup C Animalseuthanized at ~72 hours post-dose (day 30) primarily for evaluation ofneuronal necrosis (Cupric Silver staining). Subgroup D FunctionalObservation Battery (“FOB”) and learning/memory evaluations conducted onthis Subgroup. Animals euthanized at 14 days post-dose (day 41)primarily for evaluation of neuronal necrosis (Cupric Silver staining).

Histotechnology Procedures. Prior to necropsy, the rats had beenanesthetized and then subjected to intracardiac perfusion for optimalfixation. The heads from the rats in Subgroups C and D had been sent toNeuroScience Associates in Knoxville, Tenn. where the brains had beenremoved and multiply imbedded in a gelatin matrix (with 16 brains ineach block), frozen, serially sectioned at approximately 40 micrometers(through the cerebral hemispheres but not into the cerebellum), andrepresentative step sections (between 33 and 34 for each rat) stainedwith the amino cupric silver stain according to the method of de Olmoset al. (1994; Fix, 1996; Switzer, 1993, 2000).

Of the 33-34 sections present for each rat brain, approximately 17included the posterior cingulate and retrosplenial cortices.

The brains from the rats in Subgroups A and B were sent to ConsultantsIn Veterinary Pathology, Inc. where the brains were completely sliced ina standardized fashion to yield nine coronal sections, with each coronalslice being between two and three millimeters in thickness. Of thesenine coronal sections, one included the posterior cingulate cortex,while three passed through the retrosplenial cortex. Two sagittalsections of the olfactory bulbs were also prepared. The coronal brainslices were placed anterior face down (the olfactory bulbs medialsurface down) within tissue cassettes, processed to paraffin with aCitadel ® tissue processor (Shandon Lipshaw), and embedded in paraffinfollowing standardized procedures. The paraffin blocks were sectioned ata thickness of approximately 5 micrometers using a rotary microtome. Allbrain sections from the rats in Subgroups A and B were stained withhematoxylin and eosin (H&E). In addition, duplicate sections of thebrains from rats in Subgroup B were also stained with Fluoro-Jade B,using DAPI (4′,6-Diamidino-2-Phenylindole) as a counterstain. TheFluoro-Jade B procedure that was used is that reported by Schmued et al(2000). (Schmued, L. C., Hopkins, K J (2000) Brain Res. 874:123-130.Fluoro-Jade B: a high affinity fluorescent markers for the localizationof neuronal degeneration.; Schmued, L. C., Hopkins, K. J. (2000)Toxicol. Pathol. 28: 91-99. Fluoro-Jade: Novel fluorochromes fordetecting toxicant-induced neuronal degeneration.

Microscopic Evaluations. All slides were examined in “blinded” fashion.Once “unblinded” the animal identifications were entered into a PC-basedcomputer program (GLPATH; Great Laboratory ProgramS). The computerprotocol for this study was set up to include between 25 (for the cupricsilver-stained sections) and 31 (for the H&E and Fluoro-Jade B-stainedsections) representative neuroanatomic regions, these having beenselected based both on the potential for lesions to develop in theseregions and to indicate the levels of brain that were examined. All30-34 cupric silver-stained sections/rat were also examinedmicroscopically. For the Subgroup B rats, slightly different diagnoseswere used for findings made on H&E-stained sections and on those stainedwith Fluoro-Jade B. For example the term “neuron necrosis” indicates thepresence of “red dead neurons” as seen with H&E. However, neuronnecrosis within a Fluoro-Jade B-stained section would have received adiagnosis of “Fluoro-Jade Staining.”

Results

Subgroup A. The rats in Subgroup A had been euthanatized approximatelysix hours after their final subcutaneous injections. Only H&E-stainedbrain sections were examined for evidence of neuron cytoplasmicvacuolation typical of that seen following treatment with NMDA receptorantagonists such as MK-801 (Fix et al.,, 1993, 1996) (Fix, A. S., Horn,J. W., Lightman, K. A., Johnson, C. A., Long, G. G., Storts, R. W.,Farber, N. Wozniak, O. F., Olneg, J. W. (1993), Exp. Neruol. 123:204-215. Neuronal vacuolization and necrosis induced y thenon-competitive N-methyl-D-aspartate (NMDA antagonist MK(+)801,(dizocilpine maleate), a light and electron microscopic evaluation ofthe rat retrosplineal cortex.; Fix, A. S., Ross, J. F, Stitzer, S. R.Switzer, R. C. (1996) Toxicol Pathol. 24: 291-304. Integrated evaluationof the central nervous system lesions: stains for neurons, astrocytes,and inicroglia reveal the spatial an temporal features of MK-801 inducedneuronal necrosis in the rat cerbral cortex.)

This pattern of neuron vacuolation (which typically involves neuronswithin Layers II and III of the retrosplenial cortex) was not foundwithin any of the rats in this phase of the study. For the females inSubgroup B, one rat in each of Groups 1 and 2 plus two rats in Group 5(i.e. MK-801-treated) had minimal neuron necrosis considered to bewithin background frequency and to be of no biologic significance.

Subgroup B. The rats in Subgroup B had been euthanized approximately 24hours after the final injection. Both H&E and Fluoro-Jade B-stainedbrain sections were examined from these rats to detect any residualvacuolation and/or early evidence of neuronal necrosis within theposterior cingulate and retrosplenial cortices. In the male rats,minimal Fluoro-Jade staining was present within the piriform cortex ofone rat and in the tenia tecta of another rat. However, such minimalFluoro-Jade staining is within the expected background frequency.

In the female rats, both minimal to mild neuron necrosis (on H&E) andminimal to mild Fluoro-Jade staining were present within theretrosplenial cortex of either two or three rats in the MK-801-treatedgroup (Group 5). However, no such alterations were seen in rats injectedwith PMI-100.

Subgroup C. The rats in Subgroup C had been euthanized approximately 72hours after receiving their final injections. The brains from these ratshad been step-sectioned and stained by the amino cupric silver techniqueto detect the presence of neuronal degeneration within the posteriorcingulated and retrosplenial cortices, as well as elsewhere within thebrain.

Mild to moderate degrees of neuron degeneration were limited, inSubgroup C, to rats injected with MK-801 and were found most frequentlywithin the retrosplenial cortex. The degree of MK-801-associated neurondegeneration was greatest in the female rats. Mild neuron degenerationwas also found within the piriform cortex of two MK-801-treated femalerats, as was synaptic terminal degeneration within the stratum lacunosummoleculare of the hippocampus. This latter pattern of degeneration wasseen in three of four Subgroup C female rats present in treatment Group5.

Subgroup D. The rats in Subgroup D had been euthanatized 14 days afterreceiving their final injections, with the brains from these rats beingstep-sectioned and stained by the cupric silver technique to detect thepresence later stages of neuronal necrosis within the posteriorcingulate and retrosplenial cortices. No male rats in Subgroup D had anytreatment-related histologic alterations, although there were sporadicfindings of minimal degrees of neuron degeneration (i.e. only one or twoneurons) in a variety of locations but without any evidence of a doseeffect. In the female rats in Subgroup D, treatment-related lesions wereconfined to the retrosplenial cortex of MK-801-treated rats. All four ofthe female rats treated with MK-801 had mild to moderate degrees of axondegeneration within the retrosplenial cortex. While two females in Group2 had foci of minimal axon degeneration within the retrosplenial cortex,this alteration was confined to one section level, only, and probablyrepresented artifact. For the MK-801-treated females, on the other hand,the axonal staining and fragmentation was present within multiplesections throughout much of the retrosplenial cortex.

The fact that female rats in Subgroup C that were treated with MK-801had neuronal degeneration within the retrosplenial cortex but thatMK-801-treated females in Subgroup D only had axonal degeneration inthis region suggests that the somas of the necrotic neurons haddisappeared over the intervening 11 day period.

Discussion

In conclusion, there is no evidence that treatment of rats with PMI-100formulations of ketamine hydrochloride and benzalkonium chloride, underthe conditions of this subacute study, resulted in any neuropathologicalterations. The only treatment-related lesions were in the rats treatedwith MK-801, with these being the classical late-stage lesions of neuronand axon degeneration within the retrosplenial cortex. Retrosplenialcortex neurons with vacuolated cytoplasm were not found within thissubacute phase of the study. Neither was there any evidence of thepattern of minimal to mild vacuolation noted in Layer I of the Group 4females necropsied six hours after receiving a single subcutaneous doseof PMI-100 (in the previously performed acute study). Finally, nointer-group differences in overall cellularity of the retrosplenialcortices were noted.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated by reference herein in their entireties for allpurposes.

We claim:
 1. A spray device for intranasal delivery of a pharmaceuticalcomposition, wherein the spray device comprises a pharmaceuticalcomposition comprising an aqueous solution containing about 10% ketaminehydrochloride and about 0.001% to about 0.2% benzalkonium chloride,wherein the composition does not cause any significant neurotoxicity,and wherein the level of neurotoxicity is comparable to sterile waterwhen administered.
 2. The spray device of claim 1, wherein thepharmaceutical composition further comprises a suitable carrier selectedfrom the group consisting of water, saline, bicarbonate, sucrose andmixtures thereof.
 3. A spray device for intranasal delivery of apharmaceutical composition, wherein the spray device comprises apharmaceutical composition comprising an aqueous solution containingabout 10% ketamine hydrochloride and about 0.002% benzalkonium chloride,wherein the composition does not cause any significant neurotoxicity,and wherein the level of neurotoxicity is comparable to sterile waterwhen administered.
 4. A spray device for intranasal delivery of apharmaceutical composition, wherein the spray device comprises apharmaceutical composition comprising an aqueous solution containingabout 0.01 mg/kg to about 1 mg/kg ketamine hydrochloride and about0.001% to about 0.2% per unit dose benzalkonium chloride, wherein thecomposition does not cause any significant neurotoxicity, and whereinthe level of neurotoxicity is comparable to sterile water whenadministered.
 5. The spray device of claim 4, wherein the pharmaceuticalcomposition further comprises a suitable carrier selected from the groupconsisting of water, saline, bicarbonate, sucrose and mixtures thereof.6. The spray device of claim 4, wherein the pharmaceutical compositionis administered nasally with 1 to 5 sprays of the device.
 7. The spraydevice of claim 6, wherein the pharmaceutical composition isadministered nasally with 2 sprays of the device.